|
| Status |
Public on Jan 31, 2024 |
| Title |
S07 |
| Sample type |
SRA |
| |
|
| Source name |
polyps from solitary juvenile polyps
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: polyps from solitary juvenile polyps
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the preparation of single-cell suspensions from human tissues, both normal colonic tissues and colonic polyps were processed using the same protocol. Briefly, following surgical resection, tissues were immediately placed in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) and transported on ice to the laboratory. The tissues were minced on ice into pieces <1 mm3 and transferred to 10 ml Hanks’ solution containing 1 mg/ml collagenase IV, 1 mg/ml hyaluronidase (Sigma‒Aldrich, St. Louis, MO, USA) and 0.5 mg/ml DNase Ⅰ supplemented with 5% FBS for 30 min at 37 °C. Next, 10 ml ice-cold RPMI 1640 medium with 5% FBS was added. Following centrifugation using a swing-out rotor at 400 × g and 4 °C for 5 min, the supernatant was decanted and discarded, and the cell pellet was resuspended in 2 ml of red blood cell lysis buffer. Following a 5-min incubation at room temperature, the samples were centrifuged (500×g, 4 °C, 5 min). The samples were then resuspended in 1 ml PBS containing 0.04% BSA and filtered through 70-μm cell strainers. The prepared cells were then counted and assessed for viability with Trypan blue using a blood cell counting chamber. The cells were then resuspended at a concentration of 1–1.5 × 106 cells/ml with a final viability of > 80% for scRNA-seq.
Single-cell suspensions were loaded as approximately 10,000 cells per chip position within 15 min of completion of cell suspension preparation using Chromium Single-Cell 3´ reagent kits v3.1. Libraries were prepared using 10X Genomics library kits and sequenced on an Illumina NovaSeq 6000 to generate 150-bp paired-end reads according to the manufacturer’s instructions
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
gene-cell unique molecular identifier (UMI) matrix
|
| Data processing |
All 10× Genomics scRNA-seq gene expression raw sequencing data were processed using CellRanger software v.6.0.0
The gene-cell unique molecular identifier (UMI) matrix was analyzed with R software and the Seurat package (version 4.3.0)
The cells in each sample were also filtered for less than 50% mitochondrial reads
All genes that were not detected in ≥3 cells were discarded
Cells with fewer than 200 genes or greater than 5000 total genes were also removed
Assembly: GRCh38
Supplementary files format and content: gene-cell unique molecular identifier (UMI) matrix
|
| |
|
| Submission date |
Oct 03, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Yongjie Liu |
| E-mail(s) |
liuyongjie15@mails.ucas.ac.cn
|
| Organization name |
Institute of Genetics and Developmental Biology
|
| Street address |
No. 1 West Beichen Road, Chaoyang District
|
| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE244542 |
Single-cell landscape of the cellular microenvironment in three different colonic polyp subtypes in children |
|
